INTENDED USE

Hardy Diagnostics Mueller Hinton Media is recommended for use in the cultivation of a wide variety of microorganisms. Mueller Hinton Agar is recommended for disk diffusion sensitivity testing of non-fastidious organisms. Mueller Hinton Broth is recommended for preparing suspensions of microorganisms for disk diffusion sensitivity testing.

SUMMARY

Mueller and Hinton developed Mueller Hinton Agar in 1941 to be a protein free medium for isolating pathogenic strains of Neisseria.(6) It was found that Mueller Hinton Agar was useful in identifying sulfonimide-resistant and responsive strains of gonococci.(9) Additionally, this media has been used in standardized antimicrobial disk susceptibility testing, as described by Bauer, Kirby, et al.(2) Barry and Fay investigated the effects of altering the depth of plated Mueller Hinton Agar on disk diffusion testing, and determined a standardized depth of approximately four millimeters to be sufficient.(1) In 1970 Dewees, et al. studied the effect of storage on Mueller Hinton Agar plates used for antimicrobial disk diffusion zone sizes. Their findings indicated commercially manufactured Mueller Hinton Agar plates were suitable for use in routine susceptibility testing.(3) In addition to the above criteria, Hardy Diagnostics Mueller Hinton Agar meets the standards of performance established by the Clinical and Laboratory Standards Institute (CLSI), M2, M100, and ISO 16782 documents.(7,13)

Mueller Hinton Media contains beef infusion, casamino acids, and starch. Starch acts as a colloid that protects against toxic material in the medium. Beef infusion and casamino acids provide energy and nutrients. Agar is added when a solidifying agent is needed. The levels of tetracycline and sulfonamide inhibitors, thymidine, thymine, magnesium, and calcium ions, are controlled so as not to interfere with susceptibility testing and to yield good growth.(11)

The Kirby-Bauer antimicrobial disk diffusion procedure is used with Mueller Hinton Agar plates. It is based on the use of an antimicrobial impregnated filter paper disk. The impregnated disk is placed on an agar surface, resulting in diffusion of the antimicrobial into the surrounding medium. Effectiveness of the antimicrobial can be shown by measuring the zone of inhibition for a pure culture of an organism.(7,10) Zone diameters established for each antimicrobial determining resistant, intermediate, and sensitive results for pathogenic microorganisms are listed in the Clinical and Laboratory Standards Institute (CLSI), Performance Standards for Antimicrobial Susceptibility Testing, and M100.(12)

Mueller Hinton Broth is the same formulation, without the added agar. It is used for the cultivation of microorganisms, and for making dilutions of organisms to be used in the Kirby-Bauer disk diffusion procedure.

STORAGE AND SHELF LIFE

Storage: Upon receipt store plates at 2-8ºC. away from direct light. Tubed media may be stored at 2-30ºC. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Consult listed references for information on specimen collection.(4) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation.

Method of Use: For Mueller Hinton Broth, inoculate as per recognized practices for the cultivation of organisms.(5,7)

INTERPRETATION OF RESULTS

Consult CLSI document M100.(12)

LIMITATIONS

The disk diffusion method should not be used for obligatory anaerobes, slow growing organisms, and capnophiles. This method was standardized for facultative organisms or rapid growing aerobes.

In vitro susceptibility does not necessarily imply in vivo effectiveness.

When using the disk diffusion method, technical human errors may compromise reliability and accuracy. The following errors are common sources encountered in the clinical microbiology laboratory, and must be watched for: improper disk storage, inoculum not properly adjusted (too light or too heavy), incubation temperature deviating from 35-37°C, use of an increased CO2 atmosphere, reading plates before or after the full 16-18 hours of incubation, transcribing errors, reader error when measuring zone diameters, deterioration of the McFarland turbidity standard, and contamination or mutation in the control strain(s).

Research shows that strains of enterococci lacking thydylate synthetase are folate dependent and may fail to grow on thymidine-thymine deficient media.(11) In addition, media containing exogenous end products of the folate pathway may affect the results of organisms tested with trimethoprim-sulfamethoxazol, as sulfonamide and trimethoprim activity may be inhibited or reduced on media containing elevated levels of thymidine and para-aminobenzoic acid (PABA) or its analogs.(11)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, antimicrobial disks, McFarland 0.5 Turbidity Standard, forceps, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.